Identification and functional characterization of the muscarinic receptor M3 in the human keratinocyte cell line HaCaT

In the epidermis muscarinic cholinergic receptors are expressed in keratinocytes during normal differentiation and in regeneration of the skin. The muscarinic receptors were implicated to regulate proliferation, migration and differentiation of keratinocytes. Therefore, we characterized the muscarinic receptor in the spontaneously immortalized human keratinocyte cell line HaCaT and studied its role in cell migration. By RT-PCR, Western blot and immunocytochemistry we show that HaCaT cells express the M3 receptor subtype. This receptor was further characterized by fluorometric measurement of calcium mobilization in cell suspensions. After muscarinic stimulation a peak reaction of intracellular calcium mobilization occurred followed by a plateau phase of extracellular influx which was reversible by atropine. In dose-response curves the effective concentrations of carbachol and acetylcholine were determined as 40 and 1 mu M, respectively. The migratory activity of HaCaT cells was studied with the phagokinetic track assay. Treatment with the muscarinic agonist carbachol had no effect on cell migration. However, carbachol diminished the EGF-induced increase in migratory activity by about 26%. Interestingly, EGF and carbachol had additive effects on calcium mobilization.